Abstract：
Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.
摘要：
平衡通路基因的相对表达以最小化通量瓶颈和代谢负担是代谢工程中的关键挑战之一。这与迭代路径特别相关，如反向β-氧化（rBOX）途径，这需要控制多个节点的通量分配，以实现目标产物的有效合成。这里,我们开发了一种基于质粒的可诱导系统，用于基因表达的正交控制（称为TriO系统），并证明了其在rBOX途径中的实用性。以即插即用的方式轻松构建TriO向量，我们同时探索了酶选择和相对表达水平的溶液空间。值得注意的是,不同的个体表达水平导致产物特异性的实质性变化,范围从无产量到所需产物理论产量的约90%的最佳性能。我们获得了6.3g/L丁酸酯的滴度，在大肠杆菌中，来自甘油的2.2g/L丁醇和4.0g/L己酸酯，超过了以前使用等效酶组合报道的最佳滴度。由于在替代终止路线和高阶迭代中观察到类似的系统行为，我们设想我们的方法广泛适用于除rBOX之外的其他迭代路径。考虑到高吞吐量，使用组合启动子和RBS库的自动化菌株构建对于许多研究人员来说仍然遥不可及，尤其是在学术界，像TriO系统这样的工具可以通过降低成本来使路径设计的测试和评估民主化，时间和基础设施要求。
